Hydrogen Peroxide Stimulates the Ca2+-activated Big-Conductance K Channels (BK) Through cGMP Signaling Pathway in Cultured Human Endothelial Cells

Abstract

We used the whole cell patch-clamp technique to examine the effect of hydrogen peroxide (H₂O₂) on the Ca⁺-activated BK channels in human endothelial cells. We confirmed the previous finding that a 200 pS BK channel activity was detected when the cell membrane potential was clamped at 50 mV. Application of H₂O₂ or adding glucose oxidase (GO) stimulated BK channels. The stimulatory effect of H₂O₂ and GO was absent in cells treated with ebselen, a scavenger of reactive oxygen species (ROS). To determine whether the stimulatory effect of H₂O₂ and GO on BK channels is the result of increasing NO production in the endothelial cells, we examined the effect of H₂O₂ and GO on BK channels in the presence of 0.1 mM L-NAME which inhibits NO synthase (NOS). Inhibition of NOS completely abolished the stimulatory effect of H₂O₂ on BK channels. In contrast, treatment of endothelial cells with D-NAME did not block the effect of H₂O₂ on BK channels. Moreover, inhibiting soluble guanylate cyclase (sGC) with ODQ mimicked the effect of L-NAME and abolished the effect of H₂O₂. Addition of 8-bromo-cGMP stimulated BK channels and further application of H₂O₂ did not increase BK channel activity in the presence of cGMP analog. The notion that the effect of H₂O₂ on BK channels was the result of stimulating NO-cGMP pathway is further indicated by the observation that inhibition of PKG with KT5823 also abolished the stimulatory effect of H₂O₂ on BK channels. We conclude that H₂O₂ stimulates the Ca⁺ BK channels through NO/sGC/cGMP pathway in cultured human endothelial cells.